Long term goals. Our goals are to understand the mechanisms that regulate the concentrations of the endogenous ligands of the brain cannabinoid receptor (CB1) available to activate the receptor. This is important because of the importance of CB1 receptor activation in the regulation of signaling in the brain. Hypothesis: We hypothesize that changes in neuronal activity induce a rapid redistribution of the endocannabinoids (eCBs) from intracellular to extracellular domains. We further hypothesize that this redistribution occurs as a result of a combination of the mobilization of the eCBs from intracellular sequestration sites, increased synthesis and reduced catabolism. We also hypothesize that the primary sites of eCB catabolism are presynaptic, CB1 receptor expressing neurons. Aims: Our first aim is to determine regulation of eCB release in response to increased synaptic activity; the second is to study the mechanisms of inactivation of 2-AG; the third aim is to determine whether sterol carrier protein 2 is involved in the accumulation or sequestration of the eCBs by neurons. Methods: We will carry out these studies using cells in culture and cerebellar slices. We will use liquid chromatography:mass spectrometry to measure the effects of neuronal activation on the redistribution of the eCBs between tissue and buffer. Significance: It is becoming increasingly clear that the eCB/CB1 signaling pair plays important roles in many behavioral and neuropsychiatric disorders. In particular, several studies in both animals and humans suggest that altered eCB production could enhance the addictive liability of drugs of abuse. A CB1 receptor antagonist is being considered as a therapy for smoking cessation, for example. Therefore, increased understanding of the processes involved in the endogenous activation of the CB1 receptor will aid in our understanding of the potential for this drug to treat addiction. Novelty: Our laboratory has focused on the use of liquid chromatography mass spectrometry to assay endocannabinoids. We have coupled this technique with our ability to culture cells from the cerebellum as a unique approach to these goals. [unreadable] [unreadable] [unreadable]